Background: Hypertension (HBP) often occurs together with hypertriglyceridemia which indicates elevated triglyceride (TG) and remnant cholesterol (RC) levels. Non-fasting  blood lipid test after a daily meal has been recommended by the European Atherosclerosis Society (EAS). However, little is known about the difference between fasting and non-fasting cut-off values in assessing high TG (HTG) and high RC (HRC) in HBP outpatients. Methods: 225 Chinese outpatients with HBP, including 119 fasting patients  and 106 non-fasting patients  were enrolled in this study. Non-fasting levels of blood lipids at 2 h after a daily breakfast were also tested in 33 patients among the fasting group. Venous blood samples were collected. Results: The non-fasting group had significantly higher levels of TG and RC while lower levels of total cholesterol, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol than the fasting group (p < 0.05). According to the TG and RC cut-off values of the EAS, the percentages of HTG and HRC in the non-fasting group were 72.6% and 70.8%, respectively, whereas those in the fasting group were 57.1% and 52.9%, respectively. According to the cut-off value of marked HTG commonly used in the Chinese population in clinical practice, the percentage of marked HTG in the non-fasting group was 57.5%, whereas that in the fasting group was 34.5%. However, the percentages of HTG (57.6% vs. 51.5%) and HRC (51.5% vs. 51.5%) marked HTG (30.3% vs. 33.3%) in the fasting state and at 2 h after a daily breakfast in 33 outpatients did not reach statistical significance. Conclusion: Non-fasting blood lipid tests could find more individuals with HTG as well as those with marked HTG among Chinese outpatients with HBP. It indicates that non-fasting blood lipid tests are worth being recommended in patients with HBP. Once hypertension (HBP) coexists with dyslipidemia , all-cauDue to the long history of fasting blood lipid tests, it is generally accepted by Chinese doctors and patients . Many stIn this study, we assessed the difference in the levels of TG or RC as well as the percentages of high TG (HTG), high RC (HRC), or marked HTG between the fasting and non-fasting outpatients with HBP. Furthermore, a questionnaire survey was also conducted to learn about the awareness and acceptance of non-fasting blood lipid tests in Chinese outpatients with HBP.Two hundred and twenty-five outpatients with primary HBP were enrolled in this study in the Outpatient Department of Cardiovascular Medicine Department, Second Xiangya Hospital, Central South University from July 2019 to March 2021. HBP was defined as a history of systolic blood pressure values ≥ 140 mm Hg and/or diastolic blood pressure values ≥ 90 mm Hg for at least 3 days ,13. According to the fasting state or not, there were 119 fasting patients  and 106 non-fasting patients  visiting the Outpatient Department. Among all patients, 97 patients accepted a questionnaire survey to learn about the awareness and acceptance of non-fasting blood lipid tests. Fasting patients were those with a fasting period of at least 8 h before the blood lipid test. Non-fasting patients were those with blood lipid tests within 4 h after the last meal. All of them were excluded from a history of secondary HBP, thyroid diseases, liver and kidney diseases, autoimmune diseases, mental diseases, cancer, or other severe medical diseases before getting involved. No one took oral hypolipidemic agents or other drugs that affected lipid metabolism. All patients continued to take antihypertensive drugs as usual and their blood pressure was monitored throughout the course. This study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University and informed consent was obtained from all participants. After at least 8 h of overnight fasting, venous blood samples were collected in 119 fasting outpatients with HBP. Moreover, venous blood samples were also collected at 2 h after a daily breakfast in 33 patients among 119 fasting outpatients according to their daily habits. 106 non-fasting patients accepted non-fasting blood lipid tests after a daily breakfast or lunch, and venous blood samples were collected within 4 h after a daily meal according to their daily habits . The serum was separated from the venous blood samples. Serum levels of TG and total cholesterol (TC) were measured by automated enzymatic assays, and those of LDL-C and high-density lipoprotein cholesterol (HDL-C) were determined by the direct method, on a HITACHI 7170A analyzer  by a laboratory technician who had no knowledge of this study. The detection kits were provided by Japan and Pure Pharmaceutical Industry Co. (Wako). Levels of RC and non-HDL-C were estimated by the following formulas, RC = TC − (HDL-C) − (LDL-C) and non-HDL-C = TC − (HDL-C), respectively .According to the European joint consensus statement from the EAS , the fasRecently, we reported that the non-fasting cut-off values to determine HTG and HRC were TG level 2.02 mmol/L  and RC lIn this study, the non-fasting cut-off values to determine HTG, HRC, and marked HTG recommended by EAS and/or reported in Chinese subjects were all selected to evaluate the percentages of HTG, HRC, and marked HTG in HBP outpatients.In order to learn about the awareness and acceptance of non-fasting blood lipid tests in outpatients with HBP, 97 patients were invited to answer two questions. Each question is a single-choice patient. Awareness evaluation referred to whether outpatients should keep a fasting or non-fasting state for blood lipid tests. There were four options, i.e., ‘fasting’, ‘non-fasting, ‘either is OK’, and ‘having no idea’. Acceptance evaluation referred to the investigation of the choice made by outpatients again after being told that both fasting and non-fasting lipid tests were available and recommended. There were four options, i.e., ‘fasting’, ‘non-fasting’, ‘either is OK’, and ‘depending on doctor’. t-test. Paired t-test was used for comparing quantitative variables between the two time points. Qualitative variables were compared using the Chi-square test for R × C. All statistical analyses were performed with SPSS version 26.0. All p values were 2-tailed, and p < 0.05 was considered statistically significant. Quantitative variables of normal distribution were expressed as mean ± standard deviation (SD), and those of non-normal distribution were analyzed statistically after logarithmic conversion. Qualitative variables were expressed as numbers and percentages. The difference of the inter-group quantitative variables was analyzed by independent-samples n = 119) and the non-fasting group (n = 106) were presented in p < 0.05).There were 225 participants aged 18 to 70 recruited in this study population, and the fasting patients  and non-fasting patients  were found in 119 (52.9%) and 106 (47.1%) among the individuals, relatively. The general characteristics of the fasting group (p < 0.05) whereas lower levels of TC, LDL-C, and non-HDL-C (p < 0.05). There was no significant difference in HDL-C levels between the two groups  visited the doctors after breakfast whereas 64 patients (60.4%) visited the doctors after lunch. Furthermore, 62 patients (58.5%) were tested at 2 h, 18 patients (17.0%) were tested at 3 h, and 26 patients (24.5%) were tested at 4 h after a daily breakfast or lunch. Among 62 patients who were tested at 2 h, 28 patients (45.2%) were tested after breakfast whereas 34 patients (54.8%) were tested after lunch. Among 18 patients who were tested at 3 h, 7 patients (38.9%) were tested after breakfast whereas 11 patients (61.1%) were tested after lunch. Among 26 patients who were tested at 4 h, 12 patients (46.2%) were tested after breakfast whereas 14 patients (53.8%) were tested after lunch . Compareo groups A–F.According to the cut-off values of HTG and HRC recommended by the EAS , the perp < 0.05, In 33 outpatients, non-fasting levels of TG and RC at 2 h after a daily meal were significantly higher than fasting levels of TG and RC  were tested at 2 h after a daily breakfast, and about 55% (n = 59) were tested later than 2 h after a daily breakfast or even after a daily lunch. It was consistent with the finding that the mean TG level in the non-fasting group was more than 3.0 mmol/L while that at 2 h in 33 outpatients was about 2.5 mmol/L. Moreover, it was reported that the average TG level after a daily lunch is higher than that at 2 h after a daily breakfast in Chinese subjects [Interestingly, the difference in the percentages of fasting and non-fasting HTG was small and insignificant in 33 outpatients with HBP. In addition to the small sample size, another cause for this phenomenon could be the difference in the time of the blood lipid test. We previously found that levels of TG and RC began to rise at 2 h and peaked at 4 h after a daily meal in Chinese patients with coronary heart disease and HBP ,15. For subjects ,27,28, wChinese outpatients had little knowledge of the concept of non-fasting blood lipid tests. Among the surveyed outpatients, less than one in five ones chose non-fasting blood lipid tests. The vast majority of them chose fasting testing, which shows that this view has been widely accepted. After brief communication and notification on non-fasting blood lipid tests, nearly half of them turned to accept non-fasting lipid tests although more than one-third of them still insisted on fasting blood lipid tests. It was worth noting that about one-third of the surveyed outpatients left the decision to receive fasting or non-fasting blood lipid test to their doctors. That is to say, the application of non-fasting blood lipid tests in those subjects depends on their doctors. It indicates that the concepts of both doctors and patients must be updated through education in order to promote non-fasting blood lipid tests in clinical practice in China. There were several limitations in this study. First, the sample size was relatively small, especially compared with large-scale population studies abroad ,10. SecoNon-fasting blood lipid tests could find more individuals with HTG as well as those with marked HTG among Chinese outpatients with HBP. It indicates that non-fasting blood lipid tests are worth being recommended in patients with HBP. Considering the fact that the percentage of HTG at 2 h after a daily breakfast seemed to be close to that in the fasting state, the postprandial time-point at 2 h could be a more suitable substitute for the fasting one than that at 4 h when the fasting TG need to be assessed in the patients who visit their doctors in the postprandial state.


A numerical approach based on shifted Jacobi-Gauss collocation method for solving mixed Volterra-Fredholm integral equations is introduced. The novel technique with shifted Jacobi-Gauss nodes is applied to reduce the mixed Volterra-Fredholm integral equations to a system of algebraic equations that has an easy solved. The present algorithm is extended to solve the one and two-dimensional mixed Volterra-Fredholm integral equations. Convergence analysis for the present method is discussed and confirmed the exponential convergence of the spectral algorithm. Various numerical examples are approached to demonstrate the powerful and accuracy of the technique. Integral equations (IEs) are regarded as one of the most important types of equations. Mixed Volterra-Fredholm integral equations (MV-F-IEs) –4 arise Various numerical techniques have been proposed for solving the one-dimensional IEs, for example –10. The Spectral methods –23 have In this paper, numerical solutions of the one and two-dimensional MV-F-IEs are obtained by means of the shifted Jacobi-Gauss collocation (SJ-G-C) scheme. The solution of a such equation is approximated as a finite expansion of shifted Jacobi polynomials (SJP) for independent variables, and then we evaluate the residual of the MV-F-IEs at the shifted Jacobi-Gauss (SJ-G) quadrature points. The novel method together with the SJ-G nodes is utilized to reduce the MV-F-IEs to a system of algebraic equations that can be easily solved. We also analyze the convergence of the present method. Numerical simulations for some MV-F-IEs are presented to demonstrate the accuracy of the method.This paper is organized as follows: In Section 2, we present a few preliminary remarks and some information about SJP. Section 3 is divided into two subsections: one for solving one-dimensional linear MV-F-IEs using a new collocation method, and the other for numerically solving one-dimensional non-linear MV-F-IEs. In Section 4, the novel algorithm is expanded to solve two-dimensional MV-F-IEs. The error analysis of the proposed algorithm is discussed in Section 5. Numerical simulations are presented in Section 6 to ensure the effectiveness and accuracy of the technique. Section 7 outlines some observations and conclusions.ρ, γ > −1, y ∈  andnth derivative (n is an intger) of nSome properties of SJP are introduced in this section. Jacobi polynomials are given by Pk+1wL12.The collBased on the SJ-G-C technique, two algorithms have derived for the numerical treatment of MV-F-IEs. The main objective of the novel technique is to create the MV-F-IEs, to generate a system of algebraic equations of the unknown coefficients and the system can be solved by Newton’s iterative technique.g(ϱ), k1 and k2 are given functions. Whilst Λ(ϱ) is unknown function.In this subsection, we utilize the spectral collocation method for solving one dimensional linear MV-F-IEs in the next form ,Λ(ϱ)=g at any value of (ϱ) in the given domain from the next equationIn next form, we outline major steps of the SJ-G-C way to solve one dimensional linear MV-F-IEs. We select the approximate solution to be of the form ΛN(ϱ)=∑jρ,γ)(ϱ).As a con)(σ))dσ.In this ρ,γ)(ϱ).As a con)(σ))dσ.In this adopting  in the ng(ϱ), k2) and k2) are given functions, whilst Λ(ϱ) is an unknown function.In this subsection, we expand the preceding technique for the numerical treatment of the next nonlinear MV-F-IEs:N + 1 of SJ-G points, which given the subsequent form:Alike steps to that provided in the preceding subsection, permit one to reproduce the problem in the form,idual of  is set tIn this section, the preceding numerical algorithms have extended for numerically solving the linear and non-linear two-dimensional MV-F-IEs. The collocation points are chosen at the SJ-G interpolation nodes. The essence of the introduced technique is to discretize the two-dimensional MV-F-IEs to create a system of algebraic equations of the unknown coefficients.g and K are a given functions, whilst Λ is an unknown function. Hence, the SJ-G-C technique will be implemented to convert the preceding two-dimensional MV-F-IEs into the system of algebraic equations.In subsection, we enlarge the analysis in N + 1) × (M + 1) of SJ-G pointsLet us extend the dependent variable in the model,,γ2)(σ).Thereforadapting  as∑i=0Mz+gIn the iN + 1) × (M + 1) algebraic equations for ai,j; i = 0, ⋯, N, j = 0, ⋯, M. Hence, the convergent solutions  and K are given functions, whilst Λ is an unknowna function.In this subsection, we enlarge the preceding technique for the numerical treating of the nonlinear next form,N + 1 of SJ-G points given the next model:aij, by utilized Newton’s iterative method for counting the approximate solution ΛN,M at any value of  in the specified domain, by means of the next equationAs a consequence of the methodology presented in the preceding subsection, we can write the problem in the following modelidual of  is set tIn this section, we restate some useful lemmas , 32 and PN: L2(I) → YN be the L2 orthogonal projection, defined by Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: N/AReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Herein, the authors offered a numerical scheme based on shifted Jacobi-Gauss collocation method for handling the mixed Volterra-Fredholm integral equations. The method transform the equation into a system of algebraic equations. The present algorithm is extended to solve the two-dimensional case. Convergence analysis for the present method is discussed. Various numerical examples are approached to demonstrate the applicability of the technique. I have read the text thoroughly, it needs a careful revision before the possibility of its potential publication in PONE, the following comments should be meticulously addressed, a point-by-point rebuttal letter is a must:1. It is not good to boost citations by citing Refs via bulk criteria, see , , . Please cite only relevant work; also use the cite package to present Refs as [1-4], not .2. The main motivation of the method should be polished within the introductory section.https://link.springer.com/book/10.1007/978-3-540-71041-7 is welcome.3. All properties in Section 2 are not new, it is not fair to do not cite a seminal text for these properties, The text of Shen et al. 2011, 4. For the two models in 3.1 and 3.2, please cite a Ref in which you picked these models from, also mention the regularity conditions on all functions parameters that ascertain the existence and uniqueness of the solutions, see for instance 10.22075/IJNAA.2015.179https://ijnao.um.ac.ir/article_25278.html;https://doi.org/10.1016/j.camwa.2019.03.011;https://doi.org/10.1007/s40314-018-0633-3. Please mention this at the beginning of Section 5.5. The convergence rate of the unknown expansion coefficients was previously studied in depth in 6. Some Equations in Page 7 do not fit with the margins of the paper, please adopt.https://doi.org/10.1007/s40065-019-0243-y.7. Compare your work with 8. Mention the platform used to write the codes as well as the specifications of the machine used to debug these codes.9. Comment on future extensions in this direction in the conclusion section.10. Proofread the whole manuscript to get rid of any typos or grammatical errors.sssReviewer #2: The paper discusses the analysis and the numerical solution of mixed Volterra-Fredholm integral equations (MV-F-IEs). The numerical approach is based on shifted Jacobi-Gauss collocation (SJ-G-C) method. The proposed technique with shifted Jacobi-Gauss (SJ-G) nodes is applied to diminish the MV-F-IEs to a system of algebraic equations that are easily solved. The present algorithm is extended to solve the two-dimensional MV-F-IEs. Convergence analysis for the present method is discussed and confirms the exponential convergence of the spectral algorithm. Various numerical examples are approached to demonstrate the power and accuracy of the technique.The work is meaningful and has sufficient novelty, although the same technique seems to be applied to other models by the co-authors. However, the analysis is technical and concrete, and the results are fascinating. The overall structure seems to be logical; definitions are given most of the time. Thus, I suggest the acceptance of the paper for publication. Before that, the authors should consider the following minor comments.For the minor comments, see the attached file.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Youssri Hassan YoussriReviewer #1: Yes: A. K. OmranReviewer #2: **********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, <quillbot-extension-portal></quillbot-extension-portal>AttachmentReview_report_PONE-D-23-02279.pdfSubmitted filename: Click here for additional data file. 8 Mar 2023Dear respected EditorWe verified certain that all tables were cited in the text, as seen below.Table 6.1 mentioned in Lines 212 and 218.Table 6.2 mentioned in Line 229.Table 6.3 mentioned in Line234.Table 6.4 mentioned in Line 240.Responses to refereesI) With respect to the fi\frst referee (Reviewer: 1):1. We carefully checked our manuscript and corrected the errors through-out the paper.2. The main motivation of the method added in the introductory sec-tion.3. We checked the references list and inserted additional references.4. We have reviewed and corrected all of the reviewer's required changes.5. All additions were highlighted in red color.II) With respect to the second referee (Reviewer: 2):The main advantages of the present study are:1. We carefully checked our manuscript and corrected the errors through-out the paper.2. The main motivation of the method added in the introductory sec-tion.3. We inserted references for the properties mentioned in Section 2.4. For the two models in 3.1 and 3.2, We inserted suitable references.https://doi.org/10.1007/s40065-019-0243-5. We compared our work with y.(ex3).6. We mentioned the platform used to write the codes as well as thespeci\fcations of the machine used to debug these codes.7. We cited only relevant work; also used the cite package.8. we inserted a Comment on future extensions in this direction in theconclusion section.9. All additions were highlighted in red color.II) With respect to the Editor:1. We carefully checked our manuscript and corrected the errors through-out the paper.2. The main motivation of the method added in the introductory sec-tion.3. We replaced "multi-dimensional" by "one- and two-dimensional"inthe title.4. We inserted a comparison with proposed high dimensional spectralmethods for the same problems in literature.5. We added references for lemmas in section 5.1.6. We checked the references list and inserted additional references.7. The reference list has been updated, and the style has been uni\fed.8. All additions were highlighted in red color.AttachmentResponses.pdfSubmitted filename: Click here for additional data file. 16 Mar 2023Spectral technique with convergence analysis for solving one and two-dimensional mixed Volterra-Fredholm integral equationPONE-D-23-02279R1Dear Dr. Abdelkawy,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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ZakyAcademic EditorPLOS ONE**********<quillbot-extension-portal></quillbot-extension-portal> 28 Mar 2023PONE-D-23-02279R1 Spectral technique with convergence analysis for solving one and two-dimensional mixed Volterra-Fredholm integral equation Dear Dr. Abdelkawy:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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We identified homocitrulline (Hcit), sarcosine (Sar) and L-tyrosine (Tyr) as features that differentiated the studied groups (one-way ANOVA with least significant difference post hoc test). The Hcit concentrations in the non-IgE-mediated FA group were significantly decreased compared with the IgE-mediated FA group (p = 0.018) and the control group (p = 0.008). In AD children with a non-IgE-mediated FA, the Tyr levels were also significantly reduced compared with the controls (p = 0.009). The mean concentration of Sar was the highest in the non-IgE-mediated FA group and the lowest in the IgE-mediated FA group (p = 0.047). Future studies should elucidate the involvement of these AAs in the molecular pathway of IgE- and non-IgE-mediated allergic responses.An IgE-mediated food allergy (FA) in atopic dermatitis (AD) children should be easily differentiated from other immune-mediated adverse effects related to food. Specific IgEs for particular protein components has provided additional diagnostic value. However, component-resolved diagnostics (CRD) has not solved all diagnostic problems either. We analysed the serum profile of 42 amino acids (AAs) in 76 AD children aged 2–60 months with an IgE-mediated FA ( Children with a food allergy (FA) constitute a heterogeneous group ,2,3,4. T2) and Immuno Solid-Phase Allergen Chip (ISAC) assay, the mechanism of FA needs to be identified. At the moment, there is no indicator that specifically distinguishes between IgE-mediated and non-IgE-mediated FAs [A milestone in the history of allergy diagnostics was the discovery and evolution of component-resolved diagnostics (CRD) . CRD hasated FAs .Currently, one of the most promising strategies for searching for new diagnostic solutions, potential biomarkers and assessing differences between people with different health status is metabolomics ,22. MetaOur aim was to determine whether the occurrence of an IgE-mediated or non-IgE-mediated FA in AD children is associated with changes in serum-free AA levels. In this study, we analysed the serum profile of 42 AAs in 76 AD children aged 2–60 months using high-performance liquid chromatography coupled with mass spectrometry (LC-MS/MS). We applied a targeted metabolomic approach. To our knowledge, this is the first paper presenting the examination of a broad panel of free AAs (both proteinogenic and non-proteinogenic) in young children with AD and FAs. The analysis of free AAs in the serum of allergic patients will contribute not only to the assessment of their diagnostic usefulness but will also help to expand the knowledge about the pathomechanisms of this condition too. AAs are key components of peptides, proteins and phospholipids . ChangesThe study was approved by the Bioethical Commission of Poznan University of Medical Sciences, Poland (decision no. 372/20), and was in accordance with the requirements of the Declaration of Helsinki. Participants were recruited from the Medical Practice of MD Joanna Matysiak in Kalisz after obtaining the written consent of their parents. The main objectives and possible benefits of the study were explained to the patients’ parents. The study involved 76 children aged 2 to 60 months with chronic symptoms of AD (L20). Some patients additionally developed a severe reaction, i.e., anaphylaxis T78.0), urticaria (L50) and/or angioedema (T78.3) following food exposure. .0, urticg for 20 min, and finally, samples were stored at −80 °C until the appropriate analysis. Parents or legal guardians of the patients were required to carefully complete the questionnaire, which helped in the inclusion or exclusion of subjects from the study. After familiarising the parents with the nature of the study, venous blood was collected from the participants. Serum was separated via centrifugation at 300× All participants underwent a full medical examination. On the basis of the medical history, in vitro sIgE tests and an open oral food challenge (OFC) test, individuals were assigned to the appropriate groups: IgE-mediated allergy, non-IgE-mediated allergy and control group. The first group consisted of 36 AD children that suffered from IgE-mediated FAs . The non-IgE-mediated allergy group constituted 15 AD pediatric individuals with non-IgE-mediated allergy symptoms , while 25 AD patients without an FA  constituted the control group. Finally, we analysed changes in the serum AA profile of 76 individuals via LC-MS/MS. We used an aTRAQ kit  to quantify 42 free AAs (proteinogenic and non-proteinogenic) in the serum of participants.We determined the concentrations of total IgE and the IgEs specific to 295 allergens  in the serum of all participants using Allergy Explorer ALEX2 . ELISA-based in vitro multiplex analysis was performed according to the manufacturer’s instructions, as previously described [Positive results of the sIgE in vivo tests were corroborated using the OFC test. Participants were selectively subjected to the OFC test based on clinical history and physical examination. Patients with detectable high concentrations of IgE specific to the suspect food and a history of anaphylaxis were not tested using the OFC test. Patients were diagnosed with non-IgE-mediated FA using an open OFC test and observation of allergic symptoms after ingestion of the suspect food ,38,39. AAt the first visit, a clinical history was obtained, a detailed physical examination was performed, and parents were informed about the risks and benefits of the OFC test. Afterwards, the suspected food allergen, e.g., peanut, was fully excluded from the children’s diet for 5 weeks. The patients’ parents were then carefully instructed on how to reintroduce previously excluded products into their diet. According to the doctor’s instructions, the suspected food allergen was administered daily, gradually increasing the doses. The extension of the children’s diet was persisted until allergy symptoms renewed or the suspected food product was fully introduced. At the next follow-up visit, a detailed medical interview and physical examination of the participants were repeated. The patients that qualified for the study group were the children diagnosed with AD. In patients with negative sIgE results, a non-IgE-mediated FA was diagnosed if the following conditions were met: a clinical history indicating delayed-type FA with a positive OFC result with a suspicious food-allergen, during which the appearance of clinical symptoms were observed in a time typical for delayed reactions . We assessed all clinical symptoms; however, due to the diagnosis of AD in our subjects, we especially observed the appearance and/or exacerbation of skin lesions in the form of atopic eczema. Chronic gastrointestinal symptoms were observed in only a few patients as additional symptoms and these were late reactions. Only after analysing the clinical history of each patient, the result of additional tests, namely, sIgE and the course of OFC , the diagnosis of non-IgE-mediated FA was made.g. A total of 10 µL of the supernatant was transferred to a clean Eppendorf tube and 40 µL of borate buffer (pH = 8.5) was added. After vortexing and centrifugation, 10 µL of the obtained mixture was labeled with 5 µL of pre-diluted reagent solution (aTRAQ Reagent Δ8). After the mixing and centrifugation step, samples were incubated for 30 min at 23 °C. In order to stop the labeling reaction, 5 mL of hydroxylamine was added, and samples were incubated for 15 min at room temperature. In the next step, 32 µL of the internal standard solution was transferred to a tube and the mixture was centrifuged. Evaporation in a vacuum concentrator  for 15 min reduced the sample volume. Finally, 20 µL of water was added to concentrated samples and mixtures were transferred to an autosampler vial. First, 10 µL of 10% sulfosalicylic acid was added to a 40 µL serum sample. Then, specimens were mixed and centrifuged under appropriate conditions, i.e., 2 min at 10,000× We measured serum-free AA concentrations using liquid chromatography–tandem mass spectrometry (LC-MS/MS), as previously described . Measurements were carried out using a 3500 triple quad mass spectrometer  coupled with a liquid chromatograph. The mass spectrometer was equipped with an electrospray ionisation (ESI) source. The AAs were separated on C18 column   with a flow rate of 0.8 mL/min using a binary gradient of water (mobile phase A) and methanol (mobile phase B), both containing 0.01% heptafluorobutyric acid and 0.1% formic acid. The gradient elution proceeded in the following way: 0 min, 2% B; 0–6 min linearly from 2 to 40% B; 6–10 min, 40% B; 10–11 min linearly from 40 to 90% B; 11–12 min, 90% B; 12–13 min linearly from 90 to 2% B; 13–18 min, 2% B. The temperature of the separation was 50 °C. The measurements were carried out in positive ionisation mode. The MS parameters were set as follows: ion spray voltage, 1500 V; entrance potential, 10 V; declustering potential, 60 V; collision energy, 30 V  and collision cell exit potential, 10 V. The temperature of the ion source was set at 600 °C, and the pressures of gas 1 and gas 2 were set to 50 psig and 50 psig, respectively. The instrument was operated in multiple reaction monitoring (MRM) mode. A full list of MRM transitions for the 42 AAs and their corresponding internal standards was provided in our previous article .p < 0.05 was statistically significant. The normality of the data distribution was checked using the Shapiro–Wilk test. To compare the data between the studied groups, one-way ANOVA with the least significant difference (LSD) post hoc test was applied. For each differentiating factor, a standard univariate receiver operating characteristic (ROC) curve was performed using the MedCalc software. The ROC curves provide an assessment of the specificity and sensitivity of the discriminant factor. The area under the curve (AUC) was calculated using DeLong’s method. A greater AUC means a better classification of subjects into one of the groups [Statistica 13.0  and MedCalc software  were used for the data analysis. We applied univariate statistical tests. In most analyses, e groups ,22.The employed LC-MS/MS method enables the determination of 42 AAs (proteinogenic and non-proteinogenic) in a single run. We detected and quantified 34 out of 42 AAs in the analysed samples . The remp = 0.026), sarcosine  and L-tyrosine  differed in the IgE-mediated allergy group, the non-IgE-mediated allergy group and the control group (p = 0.018) and the control group  (p = 0.009), (p = 0.047)  A. In chi 0.009), B. The av= 0.047) C.ROC curves were calculated to determine a cut-off value of differentiating AAs for AD children with different mechanisms of an FA and patients without an FA . In thisIn this study, we successfully analysed the serum concentrations of 34 free amino acids (AAs) using a triple quadrupole mass spectrometer coupled with a liquid chromatography instrument. Serum samples were collected from 76 pediatric patients aged 2–60 months with atopic dermatitis (AD). We compared the AA profiles of AD children diagnosed with an IgE-mediated food allergy (FA) and a non-IgE-mediated FA, as well as AD pediatric patients without FA. The applied methodology ensured the high sensitivity and specificity of measurements and enabled parallel quantification of a wide AA profile in a small sample volume. The benefits of using aTRAQ reagents with the LC-MS/MS apparatus are supported by literature data ,42. The The first AA that differentiated the population of AD children was Sar, which is an N-methyl derivative of glycine. It is found in muscles and other tissues . Sar canHcit and Tyr may be considered as potential indicators of a non-IgE-mediated FA in AD patients aged 2–60 months. The concentrations of Hcit and Tyr were significantly decreased in the non-IgE-mediated allergy group in comparison with other AD children. Hcit belongs to the class of organic compounds known as l-alpha-amino acids, which have the L-configuration of the alpha-carbon atom . It is aThe identified abnormalities in the AA profile are the basis for more advanced research and additional explanation and differentiation of the molecular mechanisms of FAs in AD children. At the moment, there is no accurate, specific indicator that differentiates IgE-mediated and non-IgE-mediated allergic responses . Mast ceThis is the first study that used aTRAQ reagents with the LC-MS/MS system to determine the concentrations of serum-free AAs in AD children with FAs. Our study was novel, and it was impossible to precisely compare the outcomes for the studied population with other researchers. Since the number of analysed samples was limited, this study had a pilot nature. However, our data provide an interesting and fresh insight into the molecular basis of the allergic reaction in AD children. Changes in the metabolome may be prognostic. According to the literature, they appear before allergen-specific antibodies . Early dAmino acids (AAs) can act as mediators of immune system activity. Changes in the serum-free AA profile may be associated with an allergic reaction and depend on its molecular pathway. In this study, we identified homocitrulline (Hcit), sarcosine (Sar) and L-tyrosine (Tyr) as differentiating features between atopic dermatitis (AD) children with and without food allergies (FAs). Future studies should investigate and elucidate the involvement of these AAs in the molecular pathway of IgE- and non-IgE-mediated allergic responses.


This study aimed to develop cardiovascular disease (CVD) risk equations for Chinese patients with newly diagnosed type 2 diabetes (T2D) to predict 10-, 20-, and 30-year of risk.Risk equations for forecasting the occurrence of CVD were developed using data from 601 patients with newly diagnosed T2D from the Da Qing IGT and Diabetes Study with a 30-year follow-up. The data were randomly assigned to a training and test data set. In the training data set, Cox proportional hazard regression was used to develop risk equations to predict CVD. Calibration was assessed by the slope and intercept of the line between predicted and observed probabilities of outcomes by quintile of risk, and discrimination was examined using Harrell’s C statistic in the test data set. Using the Sankey flow diagram to describe the change of CVD risk over time.P = 0.002), 0.89 (P = 0.027), and 0.94 (P = 0.039) for 10-, 20-, and 30-year CVDs, respectively.Over the 30-year follow-up, corresponding to a 10,395 person-year follow-up time, 355 of 601 (59%) patients developed incident CVD; the incidence of CVD in the participants was 34.2 per 1,000 person-years. Age, sex, smoking status, 2-h plasma glucose level of oral glucose tolerance test, and systolic blood pressure were independent predictors. The C statistics of discrimination for the risk equations were 0.748 , 0.696 , and 0.687  for 10-, 20-, and 30- year CVDs, respectively. The calibration statistics for the CVD risk equations of slope were 0.88 (The risk equations forecast the long-term risk of CVD in patients with newly diagnosed T2D using variables readily available in routine clinical practice. By identifying patients at high risk for long-term CVD, clinicians were able to take the required primary prevention measures.The online version contains supplementary material available at 10.1186/s13098-023-01039-4. Reducing the cardiovascular disease (CVD) burden in diabetes mellitus is a major clinical imperative that should be prioritized in order to reduce premature death, improve quality of life, reduce individual economic burdens of associated morbidities, and reduce the high cost of medical care . CVD is Several CVD risk prediction models for patients with T2D have been developed to assist clinicians in estimating patient CVD risk and tailoring management to the needs of patients. Most of these models, however, were developed using data from predominantly Caucasian participants and do not perform well when applied to Chinese patients with T2D due to the ethnic differences in the prevalence of CVD events , 4. For The design and methods used in the Da Qing IGT and Diabetes Study have been reported elsewhere –12. BrieOf the 630 newly diagnosed T2D participants who underwent baseline examination in 1986, we excluded 19 who had missing information at baseline examination, and 10 with a known history of cardiovascular disease at enrolment. Finally, 601 participants were included in the study.All participants were tracked from their enrolment to the onset of CVD. Data were collected by personal interview, clinical examination, by trained staff for living participants, while a living spouse, sibling, or child were interviewed for the deceased with standardised questionnaires for the proxy informants. Those unable to attend the hospital because of ill health or living outside of Da Qing city were examined at home, interviewed by telephone, and examined in local hospitals. Data were then verified by review of the medical records and death certificate. CVD events were defined as the first occurrence of non-fatal or fatal myocardial infarctions, sudden death, and non-fatal or fatal stroke. The earliest date of recognition of the CVD event from medical records, interviews, or the 20- and 30-year follow-up examinations was used to define the onset of CVD. First occurrence of CVD of 355 participants were reported by December 31, 2016. Moreover, we could infer the CVD status at the 10-year follow-up based on the onset date of CVD.Participants’ characteristics are shown as the mean (± standard deviation) for quantitative parameters and as a percentage for categorical variables. Descriptive statistics were compared between participants who developed CVD or never developed CVD within a 30-year follow-up. ANOVA tests and The data were randomly assigned to two subsamples of roughly equal sizes: the training dataset (n = 300) and the test dataset (n = 301). Cox proportional hazard regression with the step forward algorithm was used to select predictors at baseline for incident CVD as long as the Akaike information criterion fell by at least the number of extra parameters. Finally, the prediction model included the baseline variables of age, sex, smoking status, plasma glucose levels 2 h after the oral glucose tolerance test (2 h-PG), and SBP. All the continuous variables were naturally logarithmically transformed to improve the discrimination and calibration of the models and to minimise the influence of extreme observations.Based on Cox proportional hazard regression, the risk score was =We evaluated the ability of the risk prediction model to discriminate participants who experience a CVD event from those who do not, using an overall C statistic  in the tSankey flow diagrams  is a datP < 0.05 was set for the statistical significance level.R software version 4.1.0 were used for all statistical analyses. A 2-tailed with During the 30-year follow-up, corresponding to 10,395 person-years of follow-up time, 59% (355/601) of participants had a first CVD incident, conferring an incidence of 34.2 per 1,000 person-years. The mean age was 48.3 years (SD = 8.7), and 52.1% (313/601) participants were females. First CVDs were occurred in 107 (18%), 271 (45%), and 355 (59%) participants of 10-, 20-, and 30-year follow-ups, respectively. The baseline characteristics of people who progressed to CVD or never developed CVD within the 30-year follow-up were shown in Table e (age) + 0.22 × current smoker (1 for yes) + 0.94 × loge (OGTT 2h plasma glucose ) + 1.37 × loge (systolic blood pressure). The average survival P = 0.002), 0.89 (P = 0.027), and 0.94 (P = 0.039) for 10-, 20-, and 30-year CVDs, respectively, indicating relative goodness of fit.The multivariable-adjusted regression coefficients and hazard ratios for incident CVD events were presented in Table Through the CVD risk equations developed in this study, the CVD risk score could be calculated for every participant in 10-, 20-, and 30-year follow-ups, respectively. Based on the risk population proportion ; and thoWe repeated a similar method while processing CVD risk scores of 20- and 30-year follow-ups. The prevalence of CVD with 45% in 20-year follow-ups in this study, therefore, participants with a 20-year CVD risk score ≥ 45% were assigned to high-risk group. The CVD risk score of 20 years with thresholds < 35%, and 35–45% were assigned to the low-risk, intermediate-risk based on the risk population proportion [Based on the definitions of high-, intermediate-, and low- risk of 10-year CVD risk score, 233 participants, 238 participants, and 130 participants were assigned to 10-high-risk group, 10-intermediate-risk group, and 10-low-risk group, separately. Meanwhile, there were 380 participants in the 20-high-risk group, 88 participants in the 20-intermediate-risk group, and 133 participants in the 20-low-risk group. Moreover, 459 participants were assigned to 30-high-risk group, 64 participants were assigned to 30-intermediate-risk group, and 78 participants were assigned to 30-low-risk group. Through the number of high-risk participants, the CVD risk equations may overestimate the risk of CVD in newly diagnosed T2D population. Through the Sankey flow diagrams in Fig. We developed risk equations to predict 10-, 20-, and 30-year CVD risk in participants with newly diagnosed T2D aged 25–74 years using data commonly available in clinical practice, such as age, sex, smoking status, 2 h-PG, and SBP. To our knowledge, these are the first long-term CVD risk equations developed in China. They are based on longer follow-up data and more comprehensively capture the progression of CVD. Our risk equations are based on newly diagnosed T2D patients without previous CVD events, and the models predict the risk of CVD as a primary event.P = 0.002), 0.89 (P = 0.027), and 0.94 (P = 0.039) for 10-year CVD, 20-year CVD, and 30-year CVD, respectively. Through the Sankey flow diagram, we observed the risk scores of CVD over time and identified patients at high risk of CVD in 30 years for early prevention.In terms of model discrimination and calibration, the C statistics were 0.748 , 0.696 , and 0.687  and the calibration statistics for CVD prediction equations of slope were 0.88 (Although the Chinese Multi-provincial Cohort Study and the China-PAR project had developed 10-year CVD risk prediction models for Chinese individuals to guide the prevention of CVD , 23, theWe observed differences of 1 h-PG in baseline between participants who developed CVD and did not develop CVD. Moreover, the performance of the predictive models using 2 h-PG or 1 h-PG as a predictor was similar (Additional file 1: Table Through the performance of CVD risk models, the performance of CVD risk equations of 20-, and 30-years has declined. The possible reason being that the predictive ability of the baseline for long-term CVD was weak, and intermediate variables or drugs need to be added to improve the predictive ability. Although the performance to predict CVD risk at 20- and 30-years was moderate, the study provided a tool for long-term CVD prediction and showed the CVD risk change over time.This study has some limitations. First, we did not validate the CVD risk prediction models in an external dataset of T2D to evaluate the performance. The external dataset of T2D with a 30-year follow-up was lacking, which limited the validation of CVD risk prediction. Second, the CVD risk equation did not include lipid information because of lacking the measures of low-density lipoproteins and the 25% missing rate of triglyceride; however, the performance of the risk equation was good and may have a wider application in clinical management. Third, although the smoking status was not significant in the model, the smoking status was included in the CVD risk equation model which was very important for CVD events. Smoking status was a risk factor for CVD events in the univariate model, however, after adjusting for age and sex, smoking status was not significant risk factor for CVD events. The reason for the smoking status not being significant was that sex, age and smoking status have some degree of correlation. Future research will verify the performance of this model in an external validation set, and further promote this model for clinical use.This study developed long-term CVD risk equations for Chinese patients with newly diagnosed T2D with a 30-year follow-up. It offers a useful tool for the clinician faced with the increasing prevalence of CVD in T2D. It will aid decision-making to provide early appropriate action to decrease the risk of adverse outcomes as well as aid health service planning.Below is the link to the electronic supplementary material.Supplementary Material 1


Cancer-associated fibroblasts (CAFs) have significant tumor regulatory functions, and CAFs-derived exosomes (CAFs-Exo) released from CAFs play an important role in the progression of oral squamous cell carcinoma (OSCC). However, a lack of comprehensive molecular biological analysis leaves the regulatory mechanisms of CAFs-Exo in OSCC unclear.We used platelet derived growth factor-BB (PDGF-BB) to induce the transformation of human oral mucosa fibroblast (hOMF) into CAFs, and extracted exosomes from the supernatant of CAFs and hOMF. We validated the effect of CAFs-Exo on tumor progression by exosomes co-culture with Cal-27 and tumor-forming in nude mice. The cellular and exosomal transcriptomes were sequenced, and immune regulatory genes were screened and validated using mRNA-miRNA interaction network analysis in combination with publicly available databases.The results showed that CAFs-Exo had a stronger ability to promote OSCC proliferation and was associated with immunosuppression. We discovered that the presence of immune-related genes in CAFs-Exo may regulate the expression of PIGR, CD81, UACA, and PTTG1IP in Cal-27 by analyzing CAFs-Exo sequencing data and publicly available TCGA data. This may account for the ability of CAFs-Exo to exert immunomodulation and promote OSCC proliferation.CAFs-Exo was found to be involved in tumor immune regulation through hsa-miR-139-5p, ACTR2 and EIF6, while PIGR, CD81, UACA and PTTG1IP may be potentially effective targets for the treatment of OSCC in the future.The online version contains supplementary material available at 10.1186/s12885-023-11028-5. Oral squamous cell carcinoma (OSCC) is the most common malignancy among head and neck squamous cell carcinomas , 2. GlobCarcinoma-associated fibroblasts (CAFs) are permanently activated fibroblasts with potent tumor-modulating effects. By secreting platelet derived growth factor-BB (PDGF-BB), human oral mucosal fibroblasts (hOMF) are reprogrammed into CAFs by OSCC, and activated CAFs have significant tumorigenic effects , 7. The Exosomes contain various components such as DNA, RNA, lipids, metabolites, cytoplasm and cell surface proteins . ExosomePrevious studies have shown that exosome-mediated immune regulation is one of the important mechanisms by which CAFs promote cancer progression , 18. CAF7—3 × 109 / ml and the particle size was measured using Flow NanoAnalyzer .hOMF  were cultured in high glucose DMEM supplemented with 10% fetal bovine serum . The hOMF was treated with 30 ng/ml of PDGF-BB  for 72 h according to previous conditions  and will4 cells/well. The absorbances were measured at 450 nm in a microplate reader and the cell proliferation rate was calculated using a standard curve based on the OD value at 450 nm. A wound-healing assay was applied to detect the migratory capability of Cal-27 cells. Culture-Insert was attached to the center of the 24-well plate. Cal-27 were incubated overnight in two chambers of Culture-Insert with 100 μl of cell suspension. Culture-Insert was gently removed the next day and culture medium containing hOMF-Exo and CAFs-Exo was added in groups and incubated at 37 °C in a 5% CO2 incubator. The gaps were observed and photographed at 0 h, 6 h,12 h and 24 h, the area of scratches were calculated by Image J software (Version 2).To clarify the uptake of exosomes in oral squamous cell carcinoma, Cal-27  was co-cultured with CAFs-Exo and hOMF-Exo. PKH26  was used to label hOMF-Exo and CAFs-Exo according to the instructions. Cal-27 cells were seeded in a confocal culture dish, the labeled hOMF-Exo and CAFs-Exo were added to the culture dish and incubated for 6 h and 24 h. 4% paraformaldehyde was fixed and then rinsed in PBS. After the nuclei were stained with DAPI, images were acquired under a laser scanning confocal microscope  in a wet state. The proliferation and cytotoxicity of Cal-27 cells were detected by cell counting kit . Cal-27 were seeded in 96-well plates at a density of 1.5 × 106 Cal-27 cells were subcutaneously injected into the nude mice (n = 5 each group). Exosomes or PBS were injected every 3 d around the tumor using a microfuge for a total of 6 injections at 100 μg CAFs-Exo, 100 μg hOMF-Exo or 100 μl PBS. On day 3 after the last injection, the mice were sacrificed by cervical dislocation. Animal euthanasia is conducted with reference to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). The formation and growth of the transplanted tumors were observed, and the tumor sizes were recorded. The fixed tumor tissues was embedded in paraffin after gradient dehydration. Paraffin-embedded tissue slices, 10 μm in thickness. Then, a hematoxylin–eosin (HE) staining kit  was used for HE staining. A portion of paraffin sections were left for immunohistochemical staining. After incubation with EDTA buffer (pH 8.0) to repair antigen (95–100 °C for 20 min), the sections were incubated with anti-CD3 or anti-CD8 in 1% BSA for 1 h at room temperature. Diaminobenzidine color development and hematoxylin counterstaining were performed, coverslips were subjected to ethanol dehydration, xylene transparency, and mounting. All glass slides were digitally scanned and semiquantitative analysis was performed using Image J software (Version 2).BALB/c-Nude mice were purchased from the Animal Experiment Center of Kunming Medical University. Mice were housed under standard conditions . All experimental protocols were approved by the Animal Experimentation Ethics Review Committee of Kunming Medical University (NO. KMMU2021750) and conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Fifteen half-male and half-female mice were randomly divided into three groups: five in the normal control (NC) group, five in the hOMF-Exo group, and five in the CAFs-Exo group. 2 × 10CAFs-Exo total protein, hOMF-Exo total protein and Cal-27 total protein were extracted using RIPA buffer with PMSF . Protein concentration was quantified by BCA protein assay . The protein samples were subjected to SDS/PAGE electrophoresis and transferred on to PVDF membranes. After blocked with 5% skim milk, the blots were probed with primary antibodies. Primary antibodies included CD9 , CD63 , CD81 , HSP90 , TSG101 , β-microtubulin , fibroblast activator protein-α , α-smooth muscle actin . After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody in TBST at room temperature for 1 h, enhanced chemiluminescence reagents  were used to detect interactions. Blots were imaged with the Chemidoc Imager  and semiquantitative analysis was performed using Image J software (Version 2).−ΔΔCT method was used to calculate the relative gene expression. The primers were synthesized by Sangon Biotech, Co., Ltd  following the protocol provided by the manufacturer. Small RNAs were reverse transcribed using the miRcute miRNA first-strand cDNA synthesis kit . The mRNA template was reversely transcribed into cDNA using reverse transcriptase Kit . Quantitative real‐time PCR (RT-qPCR) were conducted using the SuperReal PreMix Plus SYBR Green  on QuantStudio™ Real-Time PCR System . The 2t test was used to identify miRNAs that were differentially expressed between CAFs and hOMF. The screening criteria were |fold change|≥ 1.5 and FDR < 0.05.The miRNAs and mRNA of hOMF-Exo and CAFs-Exo (three replicates for each group) were extracted and sequenced with DNBseq platform, with an average yield of 35.14 Mdata per sample. The average alignment ratio of the sample comparison genome was 98.68%. Differentially expressed miRNAs and mRNA were screened with the DEGseq package of R-Studio (Version 2022.11.0). The The total RNA of Cal-27 cells treated with PBS, hOMF-Exo or CAFs-Exo (three replicates for each group), were extracted and sequenced with BGISEQ-500 platform, averagely generating about 6.60G Gb bases per sample. HISAT was used to align the clean reads to the reference genome. Bowtie 2 to align the clean reads to the reference genes. Differentially expressed mRNA were screened with the DEGseq package of R-Studio (Version 2022.11.0).t test was used to identify genes that were differentially expressed between CAFs and hOMF. Then, genes were obtained based on P-values (P < 0.05). The t test P-values were retained for subsequent analyses. Finally, the differentially expressed genes (DEGs) were labeled with immune genes. Here, the immune genes come from the GO database and include all the genes in the immune-related terms.A comprehensive method to identify potential biomarkers in OSCC exosomes was developed by integrating gene expression data. For this purpose, we first built a matrice (expression) presented as mRNA/miRNAs (row) by sample (column). RNA-seq raw read counts were converted to TPM values to scale all comparable variates and normalised across all samples. The http://string.embl.de/) is a database designed to construct a PPI network based on known and predicted PPI, and then the functional interactions between protein were analyzed. Based on the STRING online tool, the PPI of immune-related genes was constructed. Next, Cytoscape (version 3.9.1) was used to examine the potential correlation between these immune-related genes. At the same time, the MCODE application in Cytoscape was used to check the modules of the PPI network .Search Tool for the Retrieval of Interacting Genes  was used to evaluate the proportion of 22 immune cell subtypes using LM22 signatures and 1000 permutations. Cases with a CIBERSORT output of i, cj) in the dataset, referred to as Sij:i, cj) is the Pearson correlation significance P-values between the genes gi and immune cells cj.Our aim was to capture genes with relatively high co-expression in immune cells. To implement this, the first step is the normalization between the datasets using Scale function in R software. Then, we define the correlation between gene expression and each immune cell score , and used the R package named weighted gene co-expression network analysis  to construct an immune-related gene and differentially expressed miRNAs co-expression network. Firstly, Pearson correlation was used to evaluate the weighted interaction relationship between subjects in all data sets in the adjacency matrix, and abnormal samples were removed to ensure the reliability of the network construction results. Here, cutHeight was set to 7, and minSize was set to 10. Secondly, the soft-thresholding power of network construction was selected so that the constructed network accords with the power-law distribution and was closer to the real biological network state. Since the sample size was less than 20, the soft-thresholding power was determined to be 9. Then, the weighted adjacency matrix is transformed into a topological overlap measure matrix, and its connectivity in the network is estimated. The average link hierarchical clustering method is used to construct the clustering dendrogram of the topological overlap matrix (TOM). Set the smallest gene module size to 30 to obtain a suitable module, and set the threshold for merging similar modules to 0.25.The set including 369 OSCC cases were downloaded from the TCGA database. Tumor sites of oral cavity, oral tongue, buccal mucosa, lip, alveolar ridge, hard palate, and floor of mouth were included. First, we defined the sample whose age was less than or equal to 50 years old as middle younger, the sample between 50 and 75 years old as quinquagenarian, and the age greater than or equal to 75 years old as agedness. To further clarify the relationship between candidate genes expression and OSCC prognosis, the OS between different groups was compared by Kaplan–Meier analysis with the log-rank test.Here, we only focused on the immune module with the most candidate genes related to immune cells. Then, the top 30 miRNAs with the highest TOM similarity of each candidate gene in the immune module were screened, among which the miRNAs belonging to the immune module constituted the miRNAs set of candidate gene. The miRNAs that are common in all miRNAs sets are called target miRNAs. A prognostic signature was constructed using the linear combination of the expression values of the target miRNAs and their candidate genes, weighted by their estimated regression coefficients in the multivariate Cox regression analysis.https://www.targetscan.org), miRanda  and RNAhybrid databases , overlapping genes were used for mRNA-miRNA interaction network analysis and the screened target genes were considered to be associated with CAFs-Exo. The acquired target genes were used for survival analysis. The target genes were validated by RT-qPCR in cells and tumors.To investigate the target genes of CAFs-Exo in Cal-27, we analyzed the DEGs of Cal-27 after co-culture with CAFs-Exo or hOMF-Exo. In combination with TargetScan  was used for data analysis and visualization. The data provided herein are expressed as the mean ± standard deviation (SD) of at least three experimental replicates. The normality of the data was judged using the Shapiro–Wilk test. One way analysis of variance (ANOVA) was used to compare the overall mean difference between multiple groups of data, and statistically different data were then tested with least significant difference (LSD) test for differences between the two groups. For data that did not conform to a normal distribution, we used the Kruskal–Wallis test to compare the statistical significance of the groups. Our previous study demonstrated that PDGF-BB induces reprogramming of hOMF to CAFs . Using pvs CAFs-Exo, P = 0.0346; CAFs-Exo vs NC, P < 0.0001; hOMF-Exo vs NC, P = 0.0253). Using a cell co-culture system with hOMF-Exo/CAFs-Exo, the cell proliferation of Cal-27 was examined. The results showed that CAFs-Exo promoted Cal-27 cell proliferation most significantly at 72 h , indicating that CAFs-Exo promotes the proliferation of Cal-27  and total volume  were greater in the CAFs-Exo group than in the NC and hOMF-Exo groups. To clarify whether tumor proliferation was associated with the immune microenvironment, we sectioned and immunohistochemically stained tumor tissues. HE sections showed highly differentiated squamous cell carcinomas in all groups    were associated with monocyte infiltration. Among them, ACTR2, EIF6, and PECAM1 overlapped with mRNAs in the PPI module. Further analysis showed that ACTR2, EIF6, and PECAM1 were significantly associated with infiltration of at least one immune cell type (.5) Fig. C-E. TherP < 0.05, there were 162 differential miRNAs between CAFs-Exo and hOMF-Exo. We used WGCNA to analyze the exosomal mRNA and miRNA sequencing results and construct an mRNA-miRNA co-expression network. When the soft-thresholding power in WGCNA was determined as 9, three modules were identified based on the average linkage hierarchical clustering and soft threshold power . Among all modules, the blue module is considered as the immune-related module and contains 29 mRNAs and 36 miRNAs. Two candidate mRNAs, ACTR2 and EIF6, were included in the blue module.We identified the differential miRNAs of CAFs-Exo and hOMF-Exo by high-throughput sequencing. A total of 801 miRNAs were detected. When |fold change|> 1, P = 0.001). Risk scores were calculated for patients in the dataset, and the median (-0.123) was used as the cutoff value. Patients with higher risk scores had significantly shorter overall survival (OS) times compared to those with lower risk scores . ACTR2, EIF6, and hsa-miR-139-5p were analyzed with 222 DEGs using the mRNA-miRNA interaction network. The interaction network demonstrated the scale-free topology of the transcriptional regulatory network, and consisted of 93 nodes (1 miRNA and 92 mRNAs) and 164 edges , and EIF6 (P < 0.0001) and ACTR2 (P < 0.0001) were highly expressed in CAFs-Exo  and in Cal-27 cells co-cultured with CAFs-Exo (P = 0.0035), CD81 expression was significantly elevated  and in Cal-27 tumor cells co-cultured with CAFs-Exo (P < 0.0001), the expression of PIGR was significantly higher than that in the NC group. In Cal27 cells, the expression of PIGR was significantly higher in the CAFs-Exo group than in the hOMF-Exo group (P < 0.0001). Although the variability of PTTG1IP and UACA was smaller than that of CD81 and PIGR, the results were generally consistent with previous bioinformatic analyses. The above results suggest that CAFs-Exo targets and regulates oncogenes, which in turn decreases the level of immune cell infiltration in transplanted tumors and increases the growth of tumors in nude mice.We used RT-qPCR to examine the expression of relevant genes in Cal-27 cells, nude mouse tumor tissues, and exosomes to confirm the bioinformatic analysis's findings. The results showed that hsa-miR-139-5p was highly expressed in hOMF-Exo P = 0.04, and EIFOSCC is a malignant tumor of the oral epithelium that is currently treated primarily with radiotherapy and surgical resection. The tumor microenvironment mainly consists of tumor cells, infiltrating immune cells, CAFs, endothelial cells, adipocytes and other signaling molecules together, which have an important role in regulating the development of OSCC , 24. OSCThe previous studies have demonstrated that OSCC induces reprogramming of hOMF to CAFs through high secretion of PDGF-BB, and activated CAFs produce extracellular matrix and exosomes that promote OSCC growth . The hOMCAFs-Exo complete cell-to-cell communication through the exchange of miRNAs, mRNAs and lncRNAs, etc., thus contributing to the development of OSCC –30. ImmuIntegrative mRNA-miRNA network analysis is a common bioinformatic screening method used to analyze target genes –39. In tOur findings highlight that hsa-miR-139-5p and ACTR2 from CAFs-Exo may have upregulated the expression of CD81 and PIGR in the Cal-27. This may be the cause of CAFs-Exo's special OSCC proliferation-promoting ability. There were several limitations associated with the present study. Firstly, we have verified the expression of the candidate gene and the target gene. However, there is no direct evidence to prove the regulatory relationship of the candidate gene in CAFs-Exo. Secondly, CAFs-Exo obtained from the activation of hOMF by PDGF-BB can effectively interfere with the proliferation and migration of Cal-27, but other factors affecting Cal-27 such as soluble factor, apoptotic body and inflammatory body are worth further study. Thirdly, limited by the difficulty of accessing clinical specimens, our approach is to analyze the gene expression profiles of OSCC in the TCGA database to validate our results, and more mRNA and miRNA data from the same samples are still needed for validation in the future to provide more convincing results.This study shows possible crosstalk and regulation between CAFs-Exo and the immune system in OSCC. We found that miRNAs and mRNAs in CAFs-Exo play an important role in the prognosis of OSCC patients. We also reveal the potential regulatory mechanisms of CAFs-Exo released by PDGF-BB after stimulating hOMF for UACA, PTTG1IP, PIGR, CD81 in Cal-27. In the future, these results will require more data to derive and more in-vitro and in-vitro experimental validation.Additional file 1: Supplementary Fig. 1. Experimental technical roadmap. Supplementary Fig. 2. PPI network reveals several immune-related modules. (A) PPI network of differentially expressed immune-associated genes. (B-G) Six modules may play an important role in the development of OSCC. The redder the edge, the higher the co-expression, and the redder the dot, the higher the degree. ppi refers to protein interactions. Supplementary Fig. 3. Key module correlated with OSCC identified by WGCNA. (A) The heatmap depicts the TOM among all genes in the analysis. (B) Clustering of all modules. (C) Cluster dendrogram of genes. Supplementary Table 1. Primer sequences for RT-qPCR.Additional file 2.


The obtained hydrogels were further utilized as the solid electrolytes for the supercapacitors. Through a comprehensive investigation, we found that the carbonized nanospheres can act as physical crosslinking points and increase the maximum stress of the hydrogel from 0.12 to 0.31 MPa without affecting the maximum strain. In addition, the nanospaces of the carbonized nanospheres provided a pathway for ion transport, improving the capacitance of the supercapacitor from 344.83 to 369.18 mF cm−2 at 0.5 mA cm−2. The capacitance retention was also improved from 53% to 62% at 10 mA cm−2. Collectively, this study provides new insights into the application of carbonized materials to solid electrolytes.With the emergence of the energy crisis and the development of flexible electronics, there is an urgent need to develop new reliable energy supply devices with good flexibility, stable energy storage, and efficient energy transfer. Porous carbon materials have been proven to enhance the efficiency of ion transport, as the nanospaces within them serve as pathways for mass transport. However, they have been mainly investigated in the electrodes of supercapacitors and batteries. To elucidate their function in the solid electrolytes, we introduced C 60 nanospheres efficiently improve the mechanical properties and supercapacitor performance when they are added to a poly/TEMPO-cellulose hydrogel-based electrolyte.Carbonized C Supercapacitors, one of the most promising electrochemical energy storage systems, have great potential in the field of flexible power supplies due to their high power density, ultra-fast charging rate, low-cost production, and simple device structure.2–5 Unlike batteries, supercapacitors are electrochemical double-layer capacitor devices that store energy through electrostatic interactions rather than chemical reactions. Therefore, supercapacitors can stably store and quickly release energy during charge and discharge cycles.6–8 Flexible supercapacitors always require a solid electrolyte with good mechanical properties, stable electrolyte storage capacity, and firm contact with the electrode to avoid damage due to the bending or stretching of the devices during operation.9–17 Hydrogels are crosslinked polymer networks swollen in aqueous solutions. Because of the large electrolyte capacity, flexibility, and lack of leakage, hydrogels are considered as potential flexible electrolytes and have attracted more and more attention. For example, Yao et al. recently fabricated a flexible zinc-ion hybrid supercapacitor using a cellulose hydrogel electrolyte swollen with a high concentration of ZnCl2.18 By incorporating the hydrogel in a Zn anode and an activated carbon cathode (AC), the fabricated supercapacitor showed an outstanding performance . In addition, the anti-freezing property of the concentrated ZnCl2 electrolyte ensured stable operation even at −20 °C. Li and co-workers reported a conductive zwitterionic hydrogel electrolyte synthesized via copolymerization of acrylamide and zwitterionic monomer in an H2SO4/ethylene glycol aqueous solution.9 The assembled supercapacitor with activated carbon/carbon black (AC/CB) displayed a high specific capacitance of 93.5 F g−1.Recently, the rapid development of flexible electronics has attracted a great amount of attention. However, most conventional energy supplier devices lack flexibility.As in the above examples, the reported hydrogel electrolyte-based supercapacitors have shown outstanding performances, but the high cost, complicated synthesis, and use of concentrated electrolyte solutions are inevitable in the fabrication process. We now report a hydrogel electrolyte with a simple structure by introducing porous hollow carbon nanospheres into the polymer network of the hydrogel.19–21 Among various properties of the porous carbon nanospheres, the uniform size is one of the most important factors in supercapacitor performances. Smaller particle sizes and higher monodispersity of carbon nanospheres always lead to better performances because of lower mass transport and charge transfer resistances.21–23Porous carbon-based materials, especially porous carbon nanospheres, have been widely studied as electrodes for energy storage devices, such as supercapacitors and lithium-ion batteries, because they are easy and inexpensive to prepare and can facilitate the transport of various chemical species.60 is a well-known zero-dimensional molecule with the ability to self-assemble to form various morphologies, including nanospheres.24–27 The rich π-electrons, controllable morphology, and high specific surface area of C60-based materials make them applicable as nanoporous carbon electrode materials. Many reports have revealed the great potential of C60-based electrodes in energy storage devices.28,29 However, little research has been done on the effects of porous carbon nanospheres on solid electrolytes. Notably, because of the much larger size, the C60-based carbonized spheres (CS) cannot be as easily and uniformly dispersed in the hydrogel matrix as carbon dots.30 Therefore, in this work, CS was introduced into poly (PVA)/2,2,6,6-tetramethylpiperidine-1-oxyl radical-oxidized cellulose -based hydrogels via electrostatic interactions with the carboxylate groups of the TEMPO-cellulose. The obtained hydrogels showed improved mechanical properties due to the deformability of CS, which could effectively suppress the stress concentration and crack growth.31 The performance of the assembled supercapacitor was improved due to the enhanced ionic diffusion of electrolytes through the nanospace.Fullerene C60 (pC60: 99.5%) was purchased from BBS Chemicals . Isopropyl alcohol , m-xylene (99.8%), and ethylenediamine  were purchased from Wako Chemicals Corporation . 2,2,6,6-Tetramethylpiperidine-1-oxyl radical-oxidized cellulose  was supplied from Nippon Paper Industries Co., Ltd. . Poly , carbon black (CB), activated carbon (AC), and poly(vinylidene fluoride)  were obtained from Sigma-Aldrich . N-Methyl-2-pyrrolidone  was procured from Kanto Chemical Co., Inc . All chemicals were used as received.Fullerene C60-based nanospheres were synthesized as previously reported.32 A solution of EDA (0.55 mL) in m-xylene (5 mL) was prepared by applying sonication for 30 min. 5 mL of a C60 solution in m-xylene (1 mg mL−1) was then added to the EDA/m-xylene solution, and the obtained solution was thoroughly mixed on a vortex mixer for 10 s. After 40 min of incubation, the precipitates in the solution were collected by centrifugation at 9500 rpm for 10 min and repeatedly washed with IPA and DI-water 3 times, respectively. The resultant precipitates were dispersed in water/IPA (volume ratio = 5 : 1) and freeze-dried for 24 h to yield C60 nanospheres. The C60 nanospheres were then carbonized at 1100 °C in a tube furnace  under an inert nitrogen gas atmosphere to obtain the hierarchically porous hollow carbonized spheres (CS). The heating ramp, hold time, and nitrogen gas flow rate was 10 °C min−1, 3 h, and 120 cc min−1, respectively, during the carbonization process.The CThe obtained CS was added to a TC aqueous solution (1 wt%) and stirred for 12 h followed by sonication for 1 h, yielding a well-dispersed solution with 1 wt% of CS. 10% of PVA was then dissolved in the TCCS solution at 95 °C with constant stirring for 1 h. The resultant solution was centrifugated at 2000 rpm for 1 min to remove bubbles. To obtain hydrogels with good mechanical properties, the mixed solution was then frozen at −20 °C for 12 h and thawed twice.2SO4 aqueous solution (1 M) for 12 h. Every 3 h, the H2SO4 aqueous solution was replaced with a new H2SO4 aqueous solution to ensure that the water was completely replaced. The resultant hydrogel electrolyte was cut into squares about 1 cm long, 0.1 cm thick, and 0.6 g in weight to perfectly fit on the electrode surface. The working electrodes were fabricated as follows. The activated carbon (AC), conductive carbon black (CB), and poly(vinylidene fluoride) (PVDF) with a weight ratio of 8 : 1 : 1 were ground and dispersed in an appropriate amount of NMP. The dispersion was then uniformly applied on a 1 cm2 area of carbon paper and placed in a vacuum oven at 80 °C overnight to obtain the electrodes. The loading mass of active materials was around 5 mg on each electrode. A solid-state supercapacitor with a “sandwich structure” was fabricated by covering both sides of a hydrogel electrolyte with two electrodes of the same area (1 cm2) loaded with active materials. The supercapacitor was then encapsulated using a VHB tape (3 M) to maintain the integrity and prevent water absorption.The hydrogel electrolyte was prepared by immersing the hydrogel in an H−1 at 20 °C. The morphology of the CS was examined using scanning electron microscopy (SEM)  operated at 10 kV and 10 μA. The hydrogel structures were observed using SEM . The hydrogel samples were freeze-dried before the observation. Thermogravimetric analysis (TGA) was carried out on an STA 2500 Regulus Simultaneous Thermal Analysis . The differential scanning calorimetry  tests were conducted at a scanning rate of 10 °C min−1 from −80 to 20 °C under a nitrogen flow. The crystallization structures of the hydrogel samples were characterized by X-ray diffraction . The strain–stress curves of the hydrogels were measured using a testing machine  at a rate of 10 mm min−1.The chemical structures of the hydrogels were investigated by Fourier transform infrared (FTIR) spectroscopy  from 4000 to 500 cmCA, mF cm−2), energy density , and power density  of the supercapacitor are calculated according to the following equations,11CA is the areal specific capacitance, I is the constant discharge current (mA), Δt is the discharge time (s), A is the area (cm2) of an electrode, ΔV (V) is the operating voltage excluding potential drop, E is the volumetric energy density, and P is the volumetric power density.The electrochemical performance of the as-fabricated supercapacitors was measured by cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS). The electrode-specific capacitance  and large pore volume (1.346 cm3 g−1).32 Due to the lack of hydrophilic groups on the CS surface, CS could not be directly dispersed in water. To address this problem, we added CS to the TC aqueous solution. Based on the electrostatic absorption mechanism, when the neutral CS comes into contact with TC, positive charges are induced on its surface and interact with the negatively charged carboxylate groups of TC. The electrostatic interaction between TC and CS efficiently prevented the aggregation of CS, resulting in a stable and homogeneous dispersion of CS in the mixed solution.33,34 As shown in Fig. S2,The resultant Cd 200 nm . As prev32To further study the dispersion state of CS in a TC aqueous solution, the TC and TCCS aqueous solutions were freeze-dried and observed by SEM. As shown in 35,36 To rule out the effect of electron transfer on supercapacitor performances, we measured the resistivity of the freeze-dried TCCS film by applying a voltage of −1 to 1 V. As shown in Fig. S4,7 Ω cm, indicating that the TCCS network is an insulator. Due to the insulating properties of the TCCS, the PVA-TCCS hydrogel electrolyte effectively suppressed electron transfer between the two electrodes, while free ion transfer was allowed. Thus, the risk of short circuits could be significantly reduced.The CS carbonized at high temperatures always exhibits a graphitic structure and improved conductivity.−1. The TGA and DTG curves and the corresponding data are shown in Fig. S5 and Table S1,The thermal stability of TCCS was investigated by TGA and DTG. The freeze-dried TC and TCCS were heated from 20 to 1000 °C at a rate of 10 °C minThe TCCS after TGA measurement was further observed by SEM. As shown in Fig. S6,37,38 For the same reason, CS could not be dispersed in a PVA/TC aqueous solution because the carboxylate groups were occupied by PVA forming hydrogen bonds. The FTIR results also confirmed the interaction between PVA and TC. In −1. However, in the PVA-TC and PVA-TCCS hydrogels, the hydroxyl group peak significantly red-shifted to 3334 cm−1, indicating the formation of hydrogen bonds between PVA and TC.Since TCCS aqueous solution cannot form a hydrogel by freeze-thawing process, PVA was further introduced into the TCCS aqueous solution system to obtain hydrogel-based electrolytes with flexibility and good mechanical properties. As shown in −1) was much lower than that of PVA-TC (275.0 J g−1), suggesting that the CS dispersed in the hydrogel may inhibit the crystallization of the free water. A similar tendency was observed in the heating process. PVA-TCCS had a broad melting peak at 0.7 °C and a melting enthalpy of −238.8 J g−1, while PVA-TC showed a higher melting point (3.3 °C) and a higher melting enthalpy (−306.7 J g−1).DSC curves of the obtained hydrogels are displayed in 39 Compared to PVA-TC, PVA-TCCS exhibited a weak peak at 19.3° corresponding to the (101) plane of PVA. Since the carboxylate groups of TC interacted with CS, PVA chains tended to form crystals with PVA chains.40,41To investigate the crystallization of PVA in the hydrogels, XRD patterns of PVA-TC and PVA-TCCS were recorded. As shown in The morphologies of the PVA-TC and PVA-TCCS hydrogels were observed by SEM. PVA-TC  displaye31,42 In addition, CS also acted as a physical crosslinking point, improving the toughness of the resultant hydrogels. Based on both of the above properties of CS, the mechanical properties of PVA-TCCS hydrogels were effectively improved compared to PVA-TC hydrogels.As explained above, the strong electrostatic interactions between TC and CS help to build a uniform polymer network. Thus, CS could be an additive that improves the mechanical strength of the hydrogels. To investigate the effect of CS, strain–stress curves of PVA-TC and PVA-TCCS hydrogels were measured. All samples were cut into a dumbbell form of the same width, thickness, and length before measurement . As show−1, while the ionic conductivity of the PVA-TC hydrogel was 0.09 S cm−1. The higher ionic conductivity of the PVA-TCCS hydrogel is attributed to the mesoporous pores of CS, which can store a large number of charges. Due to the excellent high surface area, activated carbon is widely used in supercapacitor electrodes. The performance of the electrodes fabricated in this study was tested with a three-electrode setup: working electrode ), reference electrode (Ag/AgCl), and counter electrode (Pt). Electrochemical measurements (cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS)) were performed on the electrodes in an aqueous 1 M H2SO4 electrolyte. As shown in Fig. S9(a),x-axis at a high frequency represents the ohmic internal resistance, which was 1.1 Ω at this electrode. The rectangular shape of the CV curves shown in Fig. S9(b)−1, indicating the fast diffusion of electrolyte ions due to the presence of porous channels. Since GCD profiles of the electrodes cannot be fully recorded by our electrochemical station because of the limitation of the longest charge–discharge time, the GCD curves of the electrodes were recorded at the current densities from 2 to 10 mA cm−2 −2 at 2 mA cm−2 and reached 266.42 mF cm−2 at 10 mA cm−2. Therefore, the obtained electrodes showed a high and relatively stable performance in the liquid electrolyte, which can be further applied to the electrodes for hydrogel electrolyte-based supercapacitor devices. As shown in 43 The intercepts of the real impedance axis at high frequencies were 0.68 Ω and 0.95 Ω, corresponding to the PVA-TC and PVA-TCCS hydrogel-based devices, respectively, which represent the internal resistance of the devices. The low resistance indicated that the electrodes adheres to the electrolyte and the interface contact resistance is reduced.44Before the fabrication of the supercapacitors, the ionic conductivities of the obtained hydrogel electrolytes were measured by EIS. As presented in Fig. S8 and Table S2,−1 to 200 mV s−1 showed a rectangular shape at room temperatures, indicating that the device has a good capacitive performance.45 Compared to the PVA-TC-based device, the CV curve of the PVA-TCCS hydrogel-based device  of the TEMPO-cellulose. The oxygen-based functional groups interacted with CS in the PVA-TCCS hydrogels, being electrochemically inactive. As a result, the PVA-TCCS system exhibited an electrical double-layer charge storage mechanism.The CV curves of the supercapacitors with different scanning speeds were measured. In the electrochemical window of 1 V, the CV curve of the supercapacitor with the PVA-TC hydrogel showed a symmetrical shape like a rectangle d device  showed a1 As shown in As shown in Fig. S10(b)−2 at 0.5 mA cm−2 and decreased to 185.54 mF cm−2 at 10 mA cm−2, whereas the PVA-TCCS hydrogel-based device exhibited a larger capacitance throughout the operational current density. The PVA-TCCS hydrogel-based device had a high areal capacitance of 369.18 mF cm−2 at 0.5 mA cm−2, and the capacitance could reach 229.02 mF cm−2 at 10 mA cm−2. Therefore, the capacitance retention of the PVA-TCCS hydrogel-based device reached 62% at 10 mA cm−2, while that of the PVA-TC hydrogel-based device was only 53%. Due to the limitations in establishing the necessary interface with the electrode material, the hydrogel electrolyte-based solid-state supercapacitors have increased internal resistance and reduced charge storage properties, compared to similar liquid systems. Hence, the specific capacitance values obtained from the hydrogel-based supercapacitors were slightly lower than those obtained from the three-electrode setup with liquid electrolyte. −2 and the areal power density was 498.30 μW cm−2. As the areal power increased to 9450.47 μW cm−2, the areal energy density was still retained at 28.41 μW h cm−2. Similar to the capacitance performance, the PVA-TC hydrogel-based device showed a relatively lower energy density compared to the PVA-TCCS hydrogel-based device. Therefore, the introduction of mesoporous CS into the hydrogel-based electrolytes could effectively improve the capacitance performance by providing mesoporous pores to store a large amount of charge and promote ion diffusion in the nanospace.The specific areal capacitances calculated from the GCD curves at various current densities are shown in 60-based carbonized spheres (CS) into a PVA/TEMPO-cellulose-based hydrogel using the electrostatic interaction between the carboxylate groups of TC and CS. In addition, supercapacitors were assembled using the PVA-TC and PVA-TCCS hydrogels, prepared by a simple freeze-thaw process. Comprehensive characterization demonstrated the multiple effects of CS on the supercapacitor performance. First, the CS could act as physical crosslinking points, which help to improve the mechanical properties of the hydrogel by mitigating stress-induced crack propagation. Second, the nanospaces occupied by the CS significantly increased the ion transport efficiency. Therefore, this work provides new insights into the application of porous carbonized materials as solid electrolytes for the fabrication of energy storage devices.In this study, we introduced CHan Jia: investigation and writing – original draft; Sabina Shahi: investigation; Lok Kumar Shrestha: conceptualization, investigation and writing – review & editing; Katsuhiko Ariga: conceptualization and writing – review & editing; Tsuyoshi Michinobu: supervision, funding acquisition and writing – review & editing.There are no conflicts to declare.RA-013-D3RA03349J-s001